scholarly journals Debunking the "junk": Unraveling the role of lncRNA–miRNA–mRNA networks in fetal hemoglobin regulation

2021 ◽  
Author(s):  
Motiur Rahaman ◽  
Chiranjib Bhowmick ◽  
Jaikrishna Komanapalli ◽  
Mandrita Mukherjee ◽  
Prasanna Kumar Byram ◽  
...  
Keyword(s):  
Globin Gene ◽  
Fetal Hemoglobin ◽  
In Silico Analysis ◽  
Clinical Severity ◽  
Differentially Expressed ◽  
Supervised Machine Learning ◽  
Pharmacological Intervention ◽  
Differentially Expressed Mirnas ◽  
Hbf Induction ◽  
Globin Gene Regulation

Fetal hemoglobin (HbF) induction is considered to be a promising therapeutic strategy to ameliorate the clinical severity of β-hemoglobin disorders, and has gained a significant amount of attention in recent times. Despite the enormous efforts towards the pharmacological intervention of HbF reactivation, progress has been stymied due to limited understanding of γ-globin gene regulation. In this study, we intended to investigate the implications of lncRNA-associated competing endogenous RNA (ceRNA) interactions in HbF regulation. Probe repurposing strategies for extraction of lncRNA signatures and subsequent in silico analysis on publicly available datasets (GSE13284, GSE71935 and GSE7874) enabled us to identify 46 differentially expressed lncRNAs (DElncRNAs). Further, an optimum set of 11 lncRNAs that could distinguish between high HbF and normal conditions were predicted from these DElncRNAs using supervised machine learning and a stepwise selection model. The candidate lncRNAs were then linked with differentially expressed miRNAs and mRNAs to identify lncRNA-miRNA-mRNA ceRNA networks. The network revealed that 2 lncRNAs (UCA1 and ZEB1-AS1) and 4 miRNAs (hsa-miR-19b-3p,hsa-miR-3646,hsa-miR-937 and hsa-miR-548j) sequentially mediate cross-talk among different signaling pathways which provide novel insights into the lncRNA-mediated regulatory mechanisms, and thus lay the foundation of future studies to identify lncRNA-mediated therapeutic targets for HbF reactivation.

Genetic association studies in β-hemoglobinopathies

Hematology ◽  
2013 ◽  
Vol 2013 (1) ◽  
pp. 354-361 ◽  
Author(s):  
Swee Lay Thein
Keyword(s):  
Association Studies ◽  
Globin Gene ◽  
Genetic Association Studies ◽  
Fetal Hemoglobin ◽  
Clinical Severity ◽  
Genetic Modifiers ◽  
Genetic Studies ◽  
Globin Gene Regulation ◽  
Innate Ability

Abstract Characterization of the molecular basis of the β-thalassemias and sickle cell disease (SCD) clearly showed that individuals with the same β-globin genotypes can have extremely diverse clinical severity. Two key modifiers, an innate ability to produce fetal hemoglobin and coinheritance of α-thalassemia, both derived from family and population studies, affect the pathophysiology of both disorders at the primary level. In the past 2 decades, scientific research had applied genetic approaches to identify additional genetic modifiers. The review summarizes recent genetic studies and key genetic modifiers identified and traces the story of fetal hemoglobin genetics, which has led to an emerging network of globin gene regulation. The discoveries have provided insights on new targets for therapeutic intervention and raise possibilities of developing fetal hemoglobin predictive diagnostics for predicting disease severity in the newborn and for integration into prenatal diagnosis to better inform genetic counseling.

Identification of differentially expressed microRNAs in outgrowth embryos compared with blastocysts and non-outgrowth embryos in mice

2019 ◽  
Vol 31 (4) ◽  
pp. 645 ◽  
Author(s):  
Jihyun Kim ◽  
Jaewang Lee ◽  
Jin Hyun Jun
Keyword(s):  
Bioinformatics Analysis ◽  
Preimplantation Embryo ◽  
Expression Profiles ◽  
In Silico Analysis ◽  
Differentially Expressed ◽  
Recurrent Implantation Failure ◽  
Preimplantation Embryo Development ◽  
Mirna Expression Profiles ◽  
Differentially Expressed Mirnas ◽  
Silico Analysis

Recurrent implantation failure (RIF) is one of the main causes for the repeated failure of IVF, and the major reason for RIF is thought to be a miscommunication between the embryo and uterus. However, the exact mechanism underlying embryo–uterus cross-talk is not fully understood. The aim of the present study was to identify differentially expressed microRNAs (miRNAs) among blastocysts, non-outgrowth and outgrowth embryos in mice using microarray analysis. A bioinformatics analysis was performed to predict the potential mechanisms of implantation. The miRNA expression profiles differed significantly between non-outgrowth and outgrowth embryos. In all, 3163 miRNAs were detected in blastocysts and outgrowth embryos. Of these, 10 miRNA candidates (let-7b, miR-23a, miR-27a, miR-92a, miR-183, miR-200c, miR-291a, miR-425, miR-429 and miR-652) were identified as significant differentially expressed miRNAs of outgrowth embryos by in silico analysis. The expression of the miRNA candidates was markedly changed during preimplantation embryo development. In particular, let-7b-5p, miR-200c-3p and miR-23a-3p were significantly upregulated in outgrowth embryos compared with non-outgrowth blastocysts. Overall, differentially expressed miRNAs in outgrowth embryos compared with blastocysts and non-outgrowth embryos could be involved in embryo attachment, and interaction between the embryo proper and maternal endometrium during the implantation process.

Molecular Analysis of Alpha Hemoglobin Stabilizing Protein (AHSP) in Caucasian Patients with different Beta-Thalassemia Phenotypes.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3770-3770 ◽  
Author(s):  
M. Domenica Cappellini ◽  
Chiara Refaldi ◽  
Daniela Bignamini ◽  
Laura Zanaboni ◽  
Gemino Fiorelli
Keyword(s):  
High Performance ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Clinical Severity ◽  
Beta Thalassemia ◽  
Red Cell ◽  
Globin Genes ◽  
Nucleotide Polymorphisms ◽  
Thai Population ◽  
Globin Chains

Abstract Beta-thalassemia is a inherited hemoglobin disorder characterized by absent or reduced synthesis of the b globin chains. The pathophysiology and the severity of b-thalassemias reflect the degree of globin chain imbalance and the excess of free a globin chains that precipitate and cause oxidative damage in red cell precursors inducing their premature destruction in the bone marrow (ineffective erythropoiesis). Although the phenotype of b thalassemias can be modified by inherited factors such as different number of a globin genes or increased fetal hemoglobin production, other mechanisms appear to be involved. Recently, a protein, named alpha hemoglobin stabilizing protein (AHSP), that acts as a molecular chaperone specifically for free a globin chains, preventing their precipitation in red cell precursors, has been identified. To establish whether AHSP might have a role in modifying the clinical outcome of b thalassemias, we have analyzed the AHSP gene in 70 Caucasian b thalassaemic subjects: 26 patients with b°/b° genotype (Thalassaemia Major),24 patients with Thalassemia Intermedia (b°/b+ or b+/b+) and 20 patients with a Thalassaemia Intermedia phenotype but with only one mutation in the b globin gene, a normal a globin genotype and no other causes of anemia. In all the subjects, we have performed Denaturing High-Performance Liquid Chromatography (DHPLC) of the three exons and the direct genomic sequencing of coding and noncoding regions (~ 1.5 kb) of AHSP gene. No mutations able to modify the structure or function of AHSP have been found, however we identified eight single nucleotide polymorphisms (SNPs) spanned along the whole gene that segregate in four different aplotypes. To evaluate a possible relationship between a particular aplotype and b thalassemia severity, the allele frequency of each single aplotype in the tree groups has been established and compared to that of 33 Caucasian normal controls: no statistically significant association has been proved. Even though the loss of AHSP aggravates the b thalassaemia phenotype in mice, in Thalassemic Caucasian population the AHSP apparently doesn’t make changes in the clinical severity of b thalassemia confirming the results recently found in Thai population.

Altered Expression of Transcription and Chromatin Remodeling Factors in Deletional HPFH.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2705-2705
Author(s):  
Fernando F. Costa ◽  
Tiago G. de Andrade ◽  
Anderson F. Cunha ◽  
André Fattori ◽  
Sara T.O. Saad
Keyword(s):  
Gene Regulation ◽  
Chromatin Remodeling ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Subtractive Hybridization ◽  
Cdna Libraries ◽  
Altered Expression ◽  
Hereditary Disorders ◽  
Altered Gene ◽  
Globin Gene Regulation

Abstract Hereditary Persistence of Fetal Hemoglobin is a rare, heterogeneous and benign group of hereditary disorders with an abnormal switch from fetal to adult hemoglobin, resulting in high levels of Hb F in the adult stage. A total of six deletions related to HPFH have been described, associated with increased levels of both gamma chains. Three main hypotheses have been proposed to explain the relationship between these deletions and the non-suppression of gamma genes: the removal of competitive regions that interact with the LCR; the juxtaposition of enhancer elements; and the removal of silencers. Despite evidence to support these hypotheses, however, they are not conclusive. Recently, Xiang and cols (Abstract #1215, 2004 ASH Meeting; Blood, Volume 104, issue 11, November 16, 2004) developed a YAC construct with the whole beta-globin locus containing a deletion of approximately 83.5 Kb responsible for the HPFH-2. Unexpectedly, the gamma gene was completely silenced in the adult transgenic mice. These data suggest that other mechanisms could be involved in the increased levels of HbF in these conditions. The authors speculate that other regions upstream from the cluster may harbor this activity. We, herein, investigate the possible involvement of transcription factors, using the subtractive hybridization method to identify differentially expressed transcripts in reticulocytes from a normal subject and a HPFH-2 subject. We have identified 56 and 106 unique genes in the normal and HPFH-2 cDNA libraries, respectively. Some of these are transcription (zinc fingers and homeobox proteins) and chromatin remodeling (NAP and SWI like proteins) factors that could participate in globin gene regulation. These genes are located in cis or in trans to the deletion and their altered gene expression has been confirmed by Quantitative Real-time PCR in other two HPFH subjects. The data may present new clues about globin gene regulation, the increased expression of gamma gene in deletional HPFH and the dynamic organization of genes and chromosomes in cells.

Transcriptional Regulation of γ-Globin Gene Expression by KLF4

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 645-645
Author(s):  
Inderdeep S Kalra ◽  
Md. M Alam ◽  
Betty S Pace
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Sodium Butyrate ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
K562 Cells ◽  
Mrna Levels ◽  
Klf4 Expression ◽  
Globin Gene Regulation ◽  
Globin Gene Expression

Abstract Abstract 645 Kruppel-like factors (KLFs) are a family of Cys2His2 zinc-finger DNA binding proteins that regulate gene expression through CACCC/GC/GT box binding in various gene promoters. The CACCC element is also critical for developmental regulation of the human γ-globin and β-globin genes; therefore studies to identify transcription factors that bind the CACCC element to alter gene expression are desirable. By microarray-based gene profiling, we identified two Kruppel-like factors, KLF4 and KLF12 whose expression levels decreased simultaneously with γ-globin silencing during in vitro erythroid maturation. Subsequent reverse transcription quantitative PCR (RT-qPCR) analysis confirmed KLF4 and KLF12 mRNA levels decreased 56-fold and 16-fold respectively in erythroid progenitors from day 7 to day 28 with over 90% γ-globin gene silencing. The effects of known fetal hemoglobin inducers hemin (50μM) and sodium butyrate (2mM) on KLF factor expression was tested in K562 cells. Hemin and sodium butyrate increased KLF4 3-fold (p<0.05) and 13-fold (p<0.01) respectively while KLF12 was only induced by butyrate. Likewise, hemin treatment of KU812 leukemia cells, which actively express γ-globin and β-globin, produced a 7-fold increase in KLF4 (p<0.05) while KLF12 levels were not changed suggesting KLF4 may be directly involved in γ-globin gene regulation. To characterize its role further siRNA-mediated loss of function studies were performed in K562 cells. A 60% knockdown of KLF4 expression produced 40% attenuation of γ-globin transcription (p<0.05). To confirm this effect, rescue experiments were performed as follows: K562 cells were treated with 100nM siKLF4 alone or in combination with the pMT3-KLF4 expression vector (10 and 20μg) for 48 hrs. The 40% knockdown of γ-globin expression produced by siKLF4 was rescued to baseline levels after enforced pMT3-KLF4 expression (p<0.05). To establish whether KLF4 directly stimulates γ-globin promoter activity, we performed co-transfection of pMT3-KLF4 and the Gγ-promoter (-1500 to +36) cloned into the pGL4.17 Luc2/neo vector; a dose-dependent increase in luciferase activity (2- to 5-fold; p<0.001) was observed. Furthermore, enforced expression of pMT3-KLF4 augmented endogenous γ-globin expression 2-fold (p<0.01). Collectively, these studies suggest that KLF4 acts as a trans-activator of γ-globin gene transcription. To address the physiological relevance of these findings, studies were extended to human primary erythroid cells grown in a two-phase liquid culture system. At day 11 when γ-globin gene expression was maximal, siKLF4 treatment produced a 60% decrease in γ/β-globin mRNA levels (p<0.001). By contrast, enforced pMT3-KLF4 expression enhanced γ/β-globin 1.5-fold at day 11 and day 28 (after γ-globin silencing); HbF levels were induced 1.5-fold (p<0.05) which was demonstrated by enzyme-linked immunosorbent assay. To gain insights into the molecular mechanism of KLF4-mediated γ-globin regulation, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP) were completed. Since CREB binding protein (CBP) is known to function as a co-activator for KLF1, 4 and 13, we also tested its role in γ-globin gene regulation. EMSA performed with K562 nuclear extract and a [γ-32P] labeled γ-CACC probe (-155 to -132 relative to the γ-globin cap site) produced three DNA-protein complexes; the addition of KLF4 or CBP antibody resulted in a marked decrease in intensity of all complexes suggesting these factors bind the γ-CACC element. ChIP assay demonstrated 10-fold and 20-fold chromatin enrichment with KLF4 and CBP antibody respectively (p<0.001) confirming in vivo binding at the γ-CACC region. Lastly, co-immunoprecipitation established protein-protein interaction between KLF4 and CBP in K562 cells. Future studies will investigate the role of CBP in KLF4-mediated γ-globin regulation which will provide molecular targets for fetal hemoglobin induction and treatment of sickle cell anemia and β-thalassemia. Disclosures: No relevant conflicts of interest to declare.

Human Fetal Hemoglobin Expression Is Regulated by the Developmental Stage-Specific Repressor BCL11A

Science ◽  
2008 ◽  
Vol 322 (5909) ◽  
pp. 1839-1842 ◽  
Author(s):  
Vijay G. Sankaran ◽  
Tobias F. Menne ◽  
Jian Xu ◽  
Thomas E. Akie ◽  
Guillaume Lettre ◽  
...  
Keyword(s):  
Association Studies ◽  
Globin Gene ◽  
Genetic Association Studies ◽  
Fetal Hemoglobin ◽  
Sequence Variants ◽  
Erythroid Cells ◽  
Cell Disease ◽  
Direct Role ◽  
Globin Gene Regulation

Differences in the amount of fetal hemoglobin (HbF) that persists into adulthood affect the severity of sickle cell disease and the β-thalassemia syndromes. Genetic association studies have identified sequence variants in the gene BCL11A that influence HbF levels. Here, we examine BCL11A as a potential regulator of HbF expression. The high-HbF BCL11A genotype is associated with reduced BCL11A expression. Moreover, abundant expression of full-length forms of BCL11A is developmentally restricted to adult erythroid cells. Down-regulation of BCL11A expression in primary adult erythroid cells leads to robust HbF expression. Consistent with a direct role of BCL11A in globin gene regulation, we find that BCL11A occupies several discrete sites in the β-globin gene cluster. BCL11A emerges as a therapeutic target for reactivation of HbF in β-hemoglobin disorders.

scholarly journals Exploring epigenetic and microRNA approaches for γ-globin gene regulation

2021 ◽  
pp. 153537022110281
Author(s):  
Athena Starlard-Davenport ◽  
Ashley Fitzgerald ◽  
Betty S Pace
Keyword(s):  
Gene Regulation ◽  
Globin Gene ◽  
Single Agent ◽  
Unmet Need ◽  
Fetal Hemoglobin ◽  
Therapeutic Interventions ◽  
Globin Genes ◽  
Chromosome 11 ◽  
Globin Gene Regulation

Therapeutic interventions aimed at inducing fetal hemoglobin and reducing the concentration of sickle hemoglobin is an effective approach to ameliorating acute and chronic complications of sickle cell disease, exemplified by the long-term use of hydroxyurea. However, there remains an unmet need for the development of additional safe and effective drugs for single agent or combination therapy for individuals with β-hemoglobinopathies. Regulation of the γ-globin to β-globin switch is achieved by chromatin remodeling at the HBB locus on chromosome 11 and interactions of major DNA binding proteins, such as KLF1 and BCL11A in the proximal promoters of the globin genes. Experimental evidence also supports a role of epigenetic modifications including DNA methylation, histone acetylation/methylation, and microRNA expression in γ-globin gene silencing during development. In this review, we will critically evaluate the role of epigenetic mechanisms in γ-globin gene regulation and discuss data generated in tissue culture, pre-clinical animal models, and clinical trials to support drug development to date. The question remains whether modulation of epigenetic pathways will produce sufficient efficacy and specificity for fetal hemoglobin induction and to what extent targeting these pathways form the basis of prospects for clinical therapy.

Human Gamma Globin Gene Regulation in Knock-In Mouse Models of Anemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1779-1779
Author(s):  
Sean C. McConnell ◽  
Yongliang Huo ◽  
Shan-Run Liu ◽  
Ting-Ting Zhang ◽  
Clayton L. Ulrey ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Mouse Models ◽  
Adult Mouse ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Model Systems ◽  
Globin Genes ◽  
Human Phenotype ◽  
Globin Gene Regulation ◽  
Gene Switching

Abstract The generation of transgenic and gene targeted mouse models of human hemoglobinopathies provides valuable opportunities to test mechanisms of human globin gene regulation and experimental therapies. Yet mice do not naturally have a fetal hemoglobin, challenging our ability to adequately model the developmental onset of disease. Transgenic model systems that contain the entire human β-globin locus present obstacles to the study of human globin gene switching, including a fetal to adult globin gene switch that occurs too early in development. The generation of genetically engineered mice with a delayed human γ to β hemoglobin switch has been a major topic of interest for our laboratory. Delayed γ globin gene expression improves the clinical progression in patients as well as animal models with hemoglobinopathies. However, molecular mechanisms involved in globin gene switching are not well understood. In this study the transcriptional and epigenetic regulation of human γ to β hemoglobin switching are analyzed in novel human knock-in (KI) mouse models that complete the switch from fetal to adult hemoglobin after birth. These KI mice were generated by replacement of the adult mouse β-globin genes by homologous recombination in embryonic stem cells with a delayed switching human γ to β globin gene construct. Quantitative real-time PCR and HPLC were used to measure mouse and human embryonic, fetal, and adult globin genes through development and show that we have given the mouse a true fetal hemoglobin. Heterozygous mice express human β-like globin genes at a high level comparable to the adult mouse β globin genes. Mutations responsible for hereditary persistence of fetal hemoglobin (HPFH) in the γ globin promoter recapitulate the human phenotype in KI mice, with over 50 fold γ globin gene upregulation in adults. These HPFH KI mice also display higher γ globin levels at birth and markedly delayed γ globin gene downregulation in the weeks following birth. These studies in KI mice demonstrate that human β-like globin genes interacting with the mouse LCR are regulated in a manner similar to what is seen in humans and may be used to study the mechanisms of globin gene switching. Greater understanding of γ-globin gene regulation will be required for achieving the therapeutic goal of reactivating silenced γ-globin genes to ameliorate severe human hemoglobinopathies.

Role of Upstream cAMP Response Element (CRE) in γ-Globin Gene Regulation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4076-4076
Author(s):  
Valya Ramakrishnan ◽  
Kajari Bhattacharya ◽  
Betty Pace
Keyword(s):  
P38 Mapk ◽  
Luciferase Activity ◽  
Trichostatin A ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
K562 Cells ◽  
Mapk Signaling ◽  
Camp Response Element ◽  
Mutant Lines ◽  
Globin Gene Regulation

Abstract Abstract 4076 Poster Board III-1011 The reactivation of fetal hemoglobin has been shown to ameliorate symptoms of sickle cell disease. Previous work from our laboratory demonstrated that histone deacetylase inhibitors such as Trichostatin A and sodium butyrate activate γ-globin gene expression via p38 MAPK signaling. Using a transient luciferase genetic reporter system, the trans-factors cAMP response element binding protein 1 (CREB1) and activating transcription factor-2 (ATF-2) were shown to trans-activate the Gγ-globin CRE, located upstream at nucleotide -1222. To study this regulatory element in a native chromatin conformation, we cloned the Gγ-globin promoter (-1500 to +36) into pGL4.17 Luc2/neo to produce the pGγLuc2 reporter construct. In addition, three mutant plasmids were created by site directed mutagenesis including the -1225 G/A mutation (pGγLuc2m1), -1227 AC/TG mutations (pGγLuc2m2) and a scrambled G-CRE, 5'-TCTATGTA-3' (pGγLuc2m3S). Each reporter along with the empty vector control (pGL4.17 Luc2/neo) was transfected into K562 cells and five stable lines including KLuc2, KGγLuc2, KGγLuc2(m1), KGγLuc2(m2) and KGγLuc2(m3s) were established using the G418 selectable marker. Luciferase activity in KGγLuc2 was 700-fold higher than the promoterless Kluc2 stable line. When the G-CRE element was mutated, we observed a 70 to 80 % decrease (p<0.05) in luciferase activity in the three mutant lines. Furthermore, a loss of Gγ-promoter inducibility was observed in the mutant lines. Treatment with butyrate (2mM) or Trichostatin A (0.5μM) resulted in a 3-fold increase of luciferase activity for the wild-type KGγLuc2 line while a 5-fold loss of inducibility was observed in the mutant stable lines. These studies support a functional role for the G-CRE in γ-globin transcription. To expand our understanding of p38 MAPK signaling in G-CRE function, siRNA studies were completed. In wild-type K562 cells, p38 siRNA molecules produced a 95% decrease in target p38 MAPK mRNA levels and a concomitant 65% loss of γ-globin expression was observed. This demonstrated that p38 MAPK is required for steady-state γ-globin gene transcription. When p38 MAPK siRNA knockdown was performed in the KGγLuc2 stable line we observed 30% reduction in luciferase activity, while this effect was lost in the mutant stable lines. Additional siRNA studies will be completed in the stable lines to determine if the target trans-factors CREB1 and ATF-2 mediate the effect of p38 MAPK in the context of chromatin. Chromatin immunoprecipitation assay will also be performed to determine in vivo binding to the G-CRE. Elucidating regulatory elements involved in γ-globin gene regulation will provide novel approaches for fetal hemoglobin induction as a treatment for sickle cell disease. Disclosures: No relevant conflicts of interest to declare.

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